Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent selfdegradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn 2؉ , the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.It is vital for cells that membranes and membrane proteins retain their integrity. Malfolded and misassembled membrane proteins, produced under stressful conditions and in non-physiological situations, should receive proteolytic quality control. In Escherichia coli, FtsH, a membrane-bound and ATP-dependent zinc metalloprotease, is known to play a central role in the degradation of unstable membrane proteins (1, 2). Thus, it degrades SecY, a subunit of protein translocase (3), and Fo subunit a of proton ATPase (4) when they have failed to assemble. It also degrades YccA, the function of which is unknown (5), and some unstable cytosolic proteins (1, 2). A characteristic feature of FtsH is that it processively degrades membrane-protein substrates by recognizing their ends when they protrude sufficiently into the cytosol (6, 7). This degradation appears to be accompanied by dislocation of the substrate from the membrane, a process presumably mediated by the ATPase function of .In E. coli, only a few other membrane-integrated proteases are known: DegS and RseP (YaeL), which introduce regulated cleavage into a membrane-bound substrate (11-13), and Lep, which cleaves off a signal peptide of secretory precursor proteins (14). HtpX was first described by Kornitzer et al. (15) as a heat-inducible protein from E. coli with sequence features of a membrane protein and a metalloprotease. They showed that its expression, in a form truncated at the C-terminal, enhanced degradation of cellular proteins in puromycin-treated cells (15). Subsequently, it was also shown that the htpX gene in Xylella fastidiosa was induced by an increase in temperature (16), whereas the Streptococcus gordonii htpX was not heat-inducible (17). Disruption of S. gordonii htpX caused changes in several properties of the cell surface, although the relationship of these changes with any protease activity of HtpX was unclear (17).We rediscovered HtpX during the course of study of cellular responses to "membrane protein stress." Our results showed that ftsH disruption led to the induction of the Cpx stress response, which wa...

A covalent dimer of alamethicin Rf30 was synthesized by linking the N-termini by a disulfide bond. When the dimer peptides were added to the cis-side of a diphytanoyl PC membrane, macroscopic channel current was induced only at cis positive voltages. The single-channel recordings showed several conductance levels that were alternately stabilized. These results indicate that the dimer peptides form stable channels by N-terminal insertion like alamethicin and that most of the pores are assembled from even numbers of helices. Taking advantages of the long open duration of the dimer peptide channels, the current-voltage (I-V) relations of the single-channels were obtained by applying fast voltage ramps during the open states. The I-V relations showed rectification, such that current from the cis-side toward the trans-side is larger than that in the opposite direction. The intrinsic rectification is mainly attributed to the macro dipoles of parallel peptide helices surrounding a central pore.

Alamethicin is supposed to form helix-bundle-type channels by inserting the N terminus into bilayer lipid membranes under sufficient voltages. The N-terminal insertion has been studied with an alamethicin dimer (di-alm) N-terminally linked by a disulfide bond and by the asymmetric addition of dithiothreitol (DTT) and tetrathionate (TT) to the membrane. When di-alm was added to the cis-side membrane, it forms long-lasting channels with the lifetime tau of about 100 ms at cis-positive voltages. The lifetime was reduced to a few milliseconds by addition of DTT to the cis-side membrane, indicating that most of the channels were formed by the monomers (alm-SH) that resulted from the cleavage of the disulfide bond in di-alm. The succeeding addition of TT to the trans-side produced channels of tau=10-20 ms besides the channels of alm-SH. The results suggested that TT reacted with the N-terminal thiol group of alm-SH located at the trans-side of the membrane to alter the lifetime. The N-terminal insertion of alamethicin helices by voltage activation, therefore, was confirmed.

Self-assembled lipid membranes formed onto gold surfaces by several methods were examined in seeking membranes suitable for detecting membrane active compounds, especially, ion-channel forming peptides. Dielectric spectroscopy was used for monitoring the membrane formation and for characterizing the electrical and structural properties of the membranes. With the membrane of a thiophospholipid/phosphatidyl choline mixture, voltage-dependent conductance changes were found when the membrane was subjected to alamethicin, a channel forming peptide. The conductance changes were characteristic of the alamethicin channel formation, suggesting the feasibility of the membrane for the sensor of membrane active compounds.Keywords: solid supported membrane/bilayer lipid membrane/dielectric spectroscopy/impedance spectroscopy/alamethicin